Myometrial Smooth Muscle Cells Research Articles

Direct Effects of Inflammatory Cytokines on Mouse Uterine Contraction.

Uterine contractions signal labor onset, with elevated pro-inflammatory cytokines playing a pivotal role. Prior studies have explored their effects on prostaglandins, oxytocin, and signaling pathways, but have overlooked their direct effects on uterine contractions. Here, we aim to investigate the direct effects of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) on contractions to ascertain if they have immediate observable effects like those reported for lipopolysaccharide (LPS) and other effects. Tension recordings were used to assess the direct effects of cytokines and/or LPS on mouse uterine contractions. Calcium imaging was employed to observe calcium oscillations in cytokine-pretreated myometrial smooth muscle cells (MSMCs) in response to oxytocin. The release of inflammatory cytokines and chemokines from uterine explants after LPS and/or cytokines application was investigated using Luminex. IL-1β, IL-6, and TNF-α rapidly enhanced contractions of term pregnant mouse uterus. LPS combined with TNF-α intensified contractions compared to LPS alone, although this effect was not statistically significant in our results (p > 0.050). Pretreatment of MSMCs with IL-1β, IL-6, or TNF-α increased calcium oscillations in response to oxytocin. LPS and/or cytokine significantly stimulated the release of IL-1β, IL-6, TNF-α, Chemokine (C-X-C motif) ligand 1 (CXCL1), and monocyte chemoattractant protein-1 (MCP1) from uterine explants in vitro. Inflammatory cytokines have short-term and long-term effects on mouse uterine contractions, which together contribute to progressively stronger contractions during labor.

Endoplasmic Reticulum Stress in Myometrial Smooth Muscle Cells and Spontaneous Contraction Changes in the Uterus of Dehydroepiandrosterone-induced Polycystic Ovary Syndrome Rats.

Myometrial changes in polycystic ovary syndrome (PCOS) are poorly investigated. Thus, we aimed to investigate endoplasmic reticulum (ER) stress in myometrial smooth muscle cells and changes in spontaneous uterine contraction in PCOS. Twenty-one female Sprague-Dawley rats (21 days old) were divided into control (n = 7), vehicle (n = 7) and PCOS (n = 7) groups. While the control group was not injected subcutaneously, the vehicle group was injected subcutaneously with sesame oil (0.2 ml/day) for 20 consecutive days. The PCOS group was injected subcutaneously with dehydroepiandrosterone (6 mg/100 g/day dissolved in 0.2 ml sesame oil) for 20 consecutive days. Blood samples were collected for the measurement of follicle stimulating-hormone (FSH), luteinizing hormone (LH), testosterone (T), estradiol (E2) and glucose-regulated protein 78 (GRP78). The mRNA expression of GRP78 in the uterine tissue samples was analysed by quantitative real-time polymerase chain reaction. GRP78 protein expression was assessed by immunohistochemistry. Myometrial smooth muscle cells were examined by transmission electron microscopy. Uterine contractions were evaluated with isolated organ bath experiments. In the PCOS group, T and LH levels increased significantly, although FSH and E2 levels decreased, but this decrease was not statistically significant. Additionally, GRP78 levels increased significantly in the PCOS group. In the PCOS group, the mRNA level, immunostaining intensity of GRP78, and ER damage grade increased, but the uterine tissue calcium levels, and the frequency and amplitude of spontaneous uterine contractions decreased. The results indicated that increased ER stress in myometrial smooth muscle cells may play a causative role in the decreased spontaneous uterine contractions in PCOS.

CD38/cADPR-mediated calcium signaling in a human myometrial smooth muscle cell line, PHM1.

Cyclic ADP-ribose (cADPR) has emerged as a calcium-regulating second messenger in smooth muscle cells. CD38 protein possesses ADP-ribosyl cyclase and cADPR hydrolase activities and mediates cADPR synthesis and degradation. We have previously shown that CD38 expression is regulated by estrogen and progesterone in the myometrium. Considering hormonal regulation in gestation, the objective of the present study was to determine the role of CD38/cADPR signaling in the regulation of intracellular calcium upon contractile agonist stimulation using immortalized pregnant human myometrial (PHM1) cells. Western blot, immunofluorescence, and biochemical studies confirmed CD38 expression and the presence of ADP-ribosyl cyclase (2.6 ± 0.1 pmol/mg) and cADPR hydrolase (26.8 ± 6.8 nmoles/mg/h) activities on the PHM1 cell membrane. Oxytocin, PGF2α, and ET-1 elicited [Ca2+]i responses, and 8-Br-cADPR, a cADPR antagonist significantly attenuated agonist-induced [Ca2+]i responses between 20% and 46% in average. The findings suggest that uterine contractile agonists mediate their effects in part through CD38/cADPR signaling to increase [Ca2+]i and presumably uterine contraction. As studies in humans are limited by the availability of myometrium from healthy donors, PHM1 cells form an in vitro model to study human myometrium.

Arrangement into layers and mechanobiology of multi-cell co-culture models of the uterine wall.

Can a co-culture of three cell types mimic the in vivo layers of the uterine wall? Three protocols tested for co-culture of endometrial epithelial cells (EEC), endometrial stromal cells (ESC), and myometrial smooth muscle cells (MSMC) led to formation of the distinct layers that are characteristic of the structure of the uterine wall in vivo. We previously showed that a layer-by-layer co-culture of EEC and MSMC responded to peristaltic wall shear stresses (WSS) by increasing the polymerization of F-actin in both layers. Other studies showed that WSS induced significant cellular alterations in epithelial and endothelial cells. Human EEC and ESC cell lines and primary MSMC were co-cultured on a collagen-coated synthetic membrane in custom-designed wells. The co-culture model, created by seeding a mixture of all cells at once, was exposed to steady WSS of 0.5 dyne/cm2 for 10 and 30 min. The co-culture of the three different cells was seeded either layer-by-layer or as a mixture of all cells at once. Validation of the models was by specific immunofluorescence staining and confocal microscopy. Alterations of the cytoskeletal F-actin in response to WSS were analyzed from the 2-dimensional confocal images through the Z-stacks following a previously published algorithm. We generated three multi-cell in vitro models of the uterine wall with distinct layers of EEC, ESC, and MSMC that mimic the in vivo morphology. Exposure of the mixed seeding model to WSS induced increased polymerization of F-actin in all the three layers relative to the unexposed controls. Moreover, the increased polymerization of F-actin was higher (P-value < 0.05) when the length of exposure was increased from 10 to 30 min. Furthermore, the inner layers of ESC and MSMC, which are not in direct contact with the applied shearing fluid, also increased their F-actin polymerization. N/A. The mixed seeding co-culture model was exposed to steady WSS of one magnitude, whereas the uterus is a dynamic organ with intra-uterine peristaltic fluid motions that vary in vivo with different time-dependent magnitude. Further in vitro studies may explore the response to peristaltic WSS or other physical and/or hormonal perturbations that may mimic the spectrum of pathophysiological aspects. Numerous in vitro models were developed in order to mimic the human endometrium and endometrium-myometrium interface (EMI) region. The present co-culture models seem to be the first constructed from EEC, ESC, and MSMC on a collagen-coated synthetic membrane. These multi-cell in vitro models better represent the complex in vivo anatomy of the EMI region. The mixed seeding multi-cell in vitro model may easily be implemented in controlled studies of uterine function in reproduction and the pathogenesis of diseases. This study was supported in part by Tel Aviv University funds. All authors declare no conflict of interest.

Impact of Cyclic Strain on Elastin Synthesis in a 3D Human Myometrial Culture Model.

The synthesis and assembly of mature, organized elastic fibers remains a limitation to the clinical use of many engineered tissue replacements. There is a critical need for a more in-depth understanding of elastogenesis regulation for the advancement of methods to induce and guide production of elastic matrix structures in engineered tissues that meet the structural and functional requirements of native tissue. The dramatic increase in elastic fibers through normal pregnancy has led us to explore the potential role of mechanical stretch in combination with pregnancy levels of the steroid hormones 17β-estradiol and progesterone on elastic fiber production by human uterine myometrial smooth muscle cells in a three-dimensional (3D) culture model. Opposed to a single strain regimen, we sought to better understand how the amplitude and frequency parameters of cyclic strain influence elastic fiber production in these myometrial tissue constructs (MTC). Mechanical stretch was applied to MTC at a range of strain amplitudes (5%, 10%, and 15% at 0.5 Hz frequency) and frequencies (0.1 Hz, 0.5 Hz, 1 Hz, and constant 0 Hz at 10% amplitude), with and without pregnancy-level hormones, for 6 days. MTC were assessed for cell proliferation, matrix elastin protein content, and expression of the main elastic fiber genes, tropoelastin (ELN) and fibrillin-1 (FBN1). Significant increases in elastin protein and ELN and FBN1 mRNA were produced from samples subjected to a 0.5 Hz, 10% strain regimen, as well as samples stretched at higher amplitude (15%, 0.5 Hz) and higher frequency (1 Hz, 10%); however, no significant effects because of third-trimester mimetic hormone treatment were determined. These results establish that a minimum level of strain is required to stimulate the synthesis of elastic fiber components in our culture model and show this response can be similarly enhanced by increasing either the amplitude or frequency parameter of applied strain. Further, our results demonstrate strain alone is sufficient to stimulate elastic fiber production and suggest hormones may not be a significant factor in regulating elastin synthesis. This 3D culture model will provide a useful tool to further investigate mechanisms underlying pregnancy-induced de novo elastic fiber synthesis and assembly by uterine smooth muscle cells.

SLO2.1/NALCN Functional Complex Activity in Mouse Myometrial Smooth Muscle Cells During Pregnancy.

At the end of pregnancy, the uterus transitions from a quiescent to a highly contractile state. This is partly due to depolarization of the resting membrane potential in uterine (myometrial) smooth muscle cells (MSMCs). Experiments with human MSMCs showed that the membrane potential is regulated by a functional complex between the sodium (Na+)-activated potassium (K+) channel SLO2.1 and the Na+ Leak Channel Non-Selective (NALCN). In human MSMCs, Na+ entering through NALCN activates SLO2.1, leading to K+ efflux, membrane hyperpolarization (cells become more negative inside), and reduced contractility. Decreased SLO2.1/NALCN activity results in reduced K+ efflux, leading to membrane depolarization, Ca2+ influx via voltage-dependent calcium channels, and increased MSMC contractility. However, all of these experiments were performed with MSMCs isolated from women at term, so the role of the SLO2.1/NALCN complex early in pregnancy was speculative. To address this question here, we examined the role of the SLO2.1/NALCN complex in regulating mouse MSMC membrane potential across pregnancy. We report that Slo2.1 and Nalcn expression change along pregnancy, being more highly expressed in MSMCs from non-pregnant and early pregnant mice than in those from late-pregnant mice. Functional studies revealed that SLO2.1 channels mediate a significant portion of the K+ current in mouse MSMCs, particularly in cells from non-pregnant and early pregnant mice. Activation of SLO2.1 by Na+ influx through NALCN led to membrane hyperpolarization in MSMCs from early pregnancy but not in MSMCs from later pregnancy. Moreover, we found that the NALCN/SLO2.1 complex regulates intracellular Ca2+ responses more in MSMCs from non-pregnant and early pregnancy mice than in MSMCs from late pregnancy. Together, these findings reveal that the SLO2.1/NALCN functional complex is conserved between mouse and humans and functions throughout pregnancy. This work could open avenues for targeted pharmacological interventions in pregnancy-related complications.

Quantification of surface-localized and total oxytocin receptor in myometrial smooth muscle cells

Oxytocin acts through the oxytocin receptor (OXTR) to modulate uterine contractility. We previously identified OXTR genetic variants and showed that, in HEK293T cells, two of the OXTR protein variants localized to the cell surface less than wild-type OXTR. Here, we sought to measure OXTR in the more native human myometrial smooth muscle cell (HMSMC) line on both the cell-surface and across the whole cell, and used CRISPR editing to add an HA tag to the endogenous OXTR gene for anti-HA measurement. Quantitative flow cytometry revealed that these cells possessed 55,000 ± 3200 total OXTRs and 4900 ± 390 cell-surface OXTRs per cell. To identify any differential wild-type versus variant localization, we transiently transfected HMSMCs to exogenously express wild-type or variant OXTR with HA and green fluorescent protein tags. Total protein expression of wild-type OXTR and all tested variants were similar. However, the two variants with lower surface localization in HEK293T cells also presented lower surface localization in HMSMCs. Overall, we confirm the differential surface localization of variant OXTR in a more native cell type, and further demonstrate that the quantitative flow cytometry technique is adaptable to whole-cell measurements.

Influence of nutrition on the course of uterine fibroids

Currently, there is a negative trend in the increase in the incidence of uterine fibroids, often diagnosed at a later date, requiring more invasive laparotomic myomectomy, up to hysterectomy. It was also revealed that the patients had an aggravated premorbid background in the form of obesity and related diseases.The purpose of the study. To study domestic and foreign studies. To identify the features of the course of uterine fibroids in patients with hypersthenic and normosthenic body types.Materials and methods. Anonymous and voluntary questionnaire survey with an in-depth collection of food history, interviews of 260 patients and analysis of their case histories with uterine fibroids who underwent laparoscopic and laparotomic myomectomy were carried out, dividing them into 2 groups according to BMI.Results. In both groups, there was the following distribution according to the presence of pregnancies: 0 – 4 % and 5 %, 1 – 86 % and 84 %, 2 – 10 % and 11 % for groups I and II, respectively (p = 0.041 criterion). 25 % in group I and 31 % in group II had a history of surgery – caesarean section. The presence of inflammatory diseases of the pelvic organs in both groups was 89.3 % and 91.6 %, respectively. Also noted was a high consumption of beef and pork by 10 and 23 %, potatoes by 8 % and 13 %, white cabbage by 12 % and 11 %, bakery products by 21 % and 26 %. Low consumption of seafood and fish products by 35 % and 56 %, dairy products by 44 % and 47 %, absolutely low content of olive oil in the daily diet. The above data are presented for groups I and II, respectively. (criterion p = 0.029-0.054).Сonclusions. Obesity plays a role as a risk factor for the development of uterine fibroids, acting either through hormonal or inflammatory mechanisms. Obesity can contribute to the development of insulin resistance and hyperinsulinemia, which can directly or indirectly influence the development of fibroids by promoting the proliferation of myometrial smooth muscle cells and increasing circulating levels of ovarian hormones.

317 A 3D printed device for electrophysiological measurement of myometrial smooth muscle cell contractions in vitro

317 A 3D printed device for electrophysiological measurement of myometrial smooth muscle cell contractions in vitro

BKCa channels are involved in spontaneous and lipopolysaccharide-stimulated uterine contraction in late gestation mice†.

The large-conductance, voltage-gated, calcium (Ca2+)-activated potassium channel (BKCa) is one of the most abundant potassium channels in the myometrium. Previous work conducted by our group has identified a link between inflammation, BKCa channels and excitability of myometrial smooth muscle cells. Here, we investigate the role of BKCa channels in spontaneous and lipopolysaccharide (LPS)-stimulated uterine contraction to gain a better understanding of the relationship between the BKCa channel and uterine contraction in basal and inflammatory states. Uteri of C57BL/6J mice on gestational day 18.5 (GD18.5) were obtained and either fixed in formalin or used immediately for tension recording or isolation of primary myocytes for patch-clamp. Paraffin sections were used for immunofluorescenctdetection of BKCa and Toll-like receptor (TLR4). For tension recordings, LPS was administered to determine its effect on uterine contractions. Paxilline, a BKCa inhibitor, was used to dissect the role of BKCa in uterine contraction in basal and inflammatory states. Finally, patch-clamp recordings were performed to investigate the relationship between LPS, the BKCa channel and membrane currents in mouse myometrial smooth muscle cells (mMSMCs). We confirmed the expression of BKCa and TLR4 in the myometrium of GD18.5 mice and found that inhibiting BKCa channels with paxilline suppressed both spontaneous and LPS-stimulated uterine contractions. Furthermore, application of BKCa inhibitors (paxilline or iberiotoxin) after LPS inhibited BKCa channel activity in mMSMCs. Moreover, pretreatment with BKCa inhibitor or the TLR4 inhibitor suppressed LPS-activated BKCa currents. Our study demonstrates that BKCa channels are involved in both basal and LPS-stimulated uterine contraction in pregnant mice.

PGF2α induces a pro-labour phenotypical switch in human myometrial cells that can be inhibited with PGF2α receptor antagonists.

Preterm birth is the leading cause of infant morbidity and mortality. There has been an interest in developing prostaglandin F2α (PGF2α) antagonists as a new treatment for preterm birth, although much of the rationale for their use is based on studies in rodents where PGF2α initiates labour by regressing the corpus luteum and reducing systemic progesterone concentrations. How PGF2α antagonism would act in humans who do not have a fall in systemic progesterone remains unclear. One possibility, in addition to an acute stimulation of contractions, is a direct alteration of the myometrial smooth muscle cell state towards a pro-labour phenotype. In this study, we developed an immortalised myometrial cell line, MYLA, derived from myometrial tissue obtained from a pregnant, non-labouring patient, as well as a novel class of PGF2α receptor (FP) antagonist. We verified the functionality of the cell line by stimulation with PGF2α, resulting in Gαq-specific coupling and Ca2+ release, which were inhibited by FP antagonism. Compared to four published FP receptor antagonists, the novel FP antagonist N582707 was the most potent compound [Fmax 7.67 ± 0.63 (IC50 21.26nM), AUC 7.30 ± 0.32 (IC50 50.43nM), and frequency of Ca2+ oscillations 7.66 ± 0.41 (IC50 22.15nM)]. RNA-sequencing of the MYLA cell line at 1, 3, 6, 12, 24, and 48h post PGF2α treatment revealed a transforming phenotype from a fibroblastic to smooth muscle mRNA profile. PGF2α treatment increased the expression of MYLK, CALD1, and CNN1 as well as the pro-labour genes OXTR, IL6, and IL11, which were inhibited by FP antagonism. Concomitant with the inhibition of a smooth muscle, pro-labour transition, FP antagonism increased the expression of the fibroblast marker genes DCN, FBLN1, and PDGFRA. Our findings suggest that in addition to the well-described acute contractile effect, PGF2α transforms myometrial smooth muscle cells from a myofibroblast to a smooth muscle, pro-labour-like state and that the novel compound N582707 has the potential for prophylactic use in preterm labour management beyond its use as an acute tocolytic drug.

Elevated expression of Toll-like receptor 4 and cytokines in both serum and myometrium at term may serve as promising biomarkers for uterine activation preceding labor.

Increased inflammation and cytokine levels are considered risk factors and promoters of preterm birth (PTB). However, the regulatory mechanism of pregnancy-related inflammation remains unclear. Toll-like receptor 4 (TLR4) plays a critical role in inflammatory responses in various diseases. Therefore, our study aimed to investigate whether TLR4 is involved in the inflammatory responses during uterine activation for labor, with the goal of identifying potential biomarkers for uterine activation at term. We used flow cytometry to detect TLR4 expression on CD14+ maternal blood monocytes in the first, second, and third trimesters. ELISA was employed to measure TLR4 and cytokines levels in the maternal serum of term non-labor (TNL), term labor (TL) women and LPS induced preterm labor and PBS injected controls. TLR4siRNA was transfected into the human myometrial smooth muscle cells (HMSMCs), which were subsequently treated with IL-1β. The mRNA and protein levels of TLR4, uterine contraction-related protein connexin 43 (CX43), oxytocin receptor (OTR), MAPK/NF-κB signaling pathway, and cytokines were analyzed using qRT-PCR, western blotting, and immunohistochemistry. The study revealed TLR4 expression on CD14+ maternal blood monocytes was higher in the third trimester group compared to the first and second trimester groups (p<0.001). Maternal serum concentrations of TLR4 and cytokines were significantly higher in the TL group than the TNL group (p<0.001). TLR4, OTR, CX43, activated MAPK/NF-κB expression, and cytokines levels were upregulated in TL group, and similarly significantly higher in the LPS-induced preterm group than in the control group. Using the HMSMCs we demonstrated that TLR4siRNA transfection suppressed contractility. Interfering with TLR4 expression reduced the expression of OTR, CX43, cytokines, and MAPK/NF-κB activation. There was a significant positive relationship between TLR4 expression and the inflammatory status in the myometrium. ROC analysis indicated that TLR4 and cytokines may serve as potential biomarkers for predicting uterine activation for labor. Our data suggest that TLR4 and cytokines can act as stimulators of uterine activation for labor at term. Furthermore, the MAPK/NF-κB pathway appears to be one of the potential signaling pathways mediating TLR4's regulation of parturition initiation.

Upregulated TIMP1 facilitates and coordinates myometrial contraction by decreasing collagens and cell adhesive capacity during human labor.

Myometrial contraction is one of the key events involved in parturition. Increasing evidence suggests the importance of the extracellular matrix (ECM) in this process, in addition to the functional role of myometrial smooth muscle cells, and our previous study identified an upregulated tissue inhibitor of metalloproteinase 1 (TIMP1) in human laboring myometrium compared to nonlabor samples. This study aimed to further explore the potential role of TIMP1 in myometrial contraction. First, we confirmed increased myometrial TIMP1 levels in labor and during labor with cervical dilation using transcriptomic and proteomic analyses, followed by real-time PCR, western blotting, and immunohistochemistry. Then, a cell contraction assay was performed to verify the decreased contractility after TIMP1 knockdown in vitro. To further understand the underlying mechanism, we used RNA-sequencing analysis to reveal the upregulated genes after TIMP1 knockdown; these genes were enriched in collagen fibril organization, cell adhesion, and ECM organization. Subsequently, a human matrix metalloproteinase (MMP) array and collagen staining were performed to determine the TIMPs, MMPs and collagens in laboring and nonlabor myometrium. A real-time cell adhesion assay was used to detect cell adhesive capacity. The results showed upregulated MMP8 and MMP9, downregulated collagens, and attenuated cell adhesive capacity in laboring myometrium, while lower MMP levels and higher collagen levels and cell adhesive capacity were observed in nonlabor. Moreover, TIMP1 knockdown led to restoration of cell adhesive capacity. Together, these results indicate that upregulated TIMP1 during labor facilitates and coordinates myometrial contraction by decreasing collagen and cell adhesive capacity, which may provide effective strategies for the regulation of myometrial contraction.

Golgi Reassembly Stacking Protein 2 Modulates Myometrial Contractility during Labor by Affecting ATP Production.

The mechanism of maintaining myometrial contractions during labor remains unclear. Autophagy has been reported to be activated in laboring myometrium, along with the high expression of Golgi reassembly stacking protein 2 (GORASP2), a protein capable of regulating autophagy activation. This study aimed to investigate the role and mechanism of GORASP2 in uterine contractions during labor. Western blot confirmed the increased expression of GORASP2 in laboring myometrium. Furthermore, the knockdown of GORASP2 in primary human myometrial smooth muscle cells (hMSMCs) using siRNA resulted in reduced cell contractility. This phenomenon was independent of the contraction-associated protein and autophagy. Differential mRNAs were analyzed using RNA sequencing. Subsequently, KEGG pathway analysis identified that GORASP2 knockdown suppressed several energy metabolism pathways. Furthermore, reduced ATP levels and aerobic respiration impairment were observed in measuring the oxygen consumption rate (OCR). These findings suggest that GORASP2 is up-regulated in the myometrium during labor and modulates myometrial contractility mainly by maintaining ATP production.

Arginase 1 and L-arginine coordinate fetal lung development and the initiation of labor in mice.

Fetal development and parturition are precisely regulated processes that involve continuous crosstalk between the mother and the fetus. Our previous discovery that wild-type mice carrying steroid receptor coactivator (Src)-1 and Src-2 double-deficient fetuses exhibit impaired lung development and delayed labor, which indicates that the signals for parturition emanate from the fetus. In this study, we perform RNA sequencing and targeted metabolomics analyses of the lungs from fetal Src-1/-2 double-knockout mice and find that expression of arginase 1 (Arg1) is significantly decreased, accompanied by increased levels of the Arg1 substrate L-arginine. Knockdown of Arg1 in the lungs of fetal mice induces apoptosis of epithelial cells and dramatically delays initiation of labor. Moreover, treatment of human myometrial smooth muscle cells with L-arginine significantly inhibits spontaneous contractions by attenuating activation of NF-κB and downregulating expression of contraction-associated protein genes. Transcription factors GR and C/EBPβ increase transcription of Arg1 in an Src-1/Src-2-dependent manner. These findings provide new evidence that fetus-derived factors may play dual roles in coordinating fetal lung development and the initiation of labor.

CAMP Compartmentalisation in Human Myometrial Cells.

Preterm birth is the leading cause of childhood mortality and morbidity. A better understanding of the processes that drive the onset of human labour is essential to reduce the adverse perinatal outcomes associated with dysfunctional labour. Beta-mimetics, which activate the myometrial cyclic adenosine monophosphate (cAMP) system, successfully delay preterm labour, suggesting a key role for cAMP in the control of myometrial contractility; however, the mechanisms underpinning this regulation are incompletely understood. Here we used genetically encoded cAMP reporters to investigate cAMP signalling in human myometrial smooth muscle cells at the subcellular level. We found significant differences in the dynamics of the cAMP response in the cytosol and at the plasmalemma upon stimulation with catecholamines or prostaglandins, indicating compartment-specific handling of cAMP signals. Our analysis uncovered significant disparities in the amplitude, kinetics, and regulation of cAMP signals in primary myometrial cells obtained from pregnant donors compared with a myometrial cell line and found marked response variability between donors. We also found that in vitro passaging of primary myometrial cells had a profound impact on cAMP signalling. Our findings highlight the importance of cell model choice and culture conditions when studying cAMP signalling in myometrial cells and we provide new insights into the spatial and temporal dynamics of cAMP in the human myometrium.

Regulation of oxytocin-induced calcium transients and gene expression in engineered myometrial tissues by tissue architecture and matrix rigidity

The uterus is susceptible to benign tumors known as fibroids, which have been associated with many pregnancy complications, including preterm labor. However, the impact of fibrotic tissue remodeling on the physiology of the myometrium, the smooth muscle layer of the uterus, is poorly understood, in large part due to a lack of model systems. In this study, we engineered healthy-like and fibrotic-like myometrium by culturing human myometrial smooth muscle cells on polyacrylamide hydrogels micropatterned with fibronectin to independently tune matrix rigidity and tissue alignment, respectively. We then evaluated calcium transients in response to oxytocin stimulation. Isotropic myometrial tissues on stiff substrates (representing fibrotic myometrium) had shorter calcium transients due to shorter decay time compared to aligned myometrial tissues on soft substrates (representing healthy myometrium). Calcium transients in aligned tissues had longer response times and longer decay times than isotropic tissues, irrespective of substrate stiffness. The amplitude of calcium transients was also higher on soft substrates compared to stiff substrates, irrespective of tissue alignment. We also performed RNA sequencing to detect differentially expressed genes between healthy- and fibrotic-like tissues, which revealed that a bitter taste receptor shown to induce smooth muscle relaxation, TAS2R31, was down-regulated in fibrotic-like tissues. Finally, we measured oxytocin-induced calcium transients in response to pre-treatment with progesterone, caffeine, thrombin, and nifedipine to demonstrate applications for our model system in drug screening. Both progesterone and caffeine caused a decrease in calcium transient duration, as expected, while thrombin and nifedipine had less impact. Collectively, our engineered model of the myometrium enables new insights into myometrial mechanobiology and can be extended to identify or screen novel drug targets.

Neglected case of a huge leiomyoma in an elderly postmenopausal woman: a case report

BackgroundFibroids are benign tumors of the female reproductive tract originating from the myometrial smooth muscle cells. They are a frequent occurrence in women of childbearing age but their incidence is rare after menopause. In addition, there is a remote possibility of malignant transformation to leiomyosarcoma in less than 1% of cases.Case presentationWe hereby report a case of large fibroid in a postmenopausal Indian female with rapid growth, raising the suspicion of malignant transformation into leiomyosarcoma. Total abdominal hysterectomy with bilateral salpingo-oophorectomy was performed. Histopathology report confirmed it to be benign leiomyoma without any evidence of neoplasia.ConclusionSudden enlargement of leiomyoma in postmenopausal women should not be ignored due to possible malignant transformation and is to be dealt immediately with hysterectomy followed by histopathology.

Mioma cervical: anatomía y abordaje quirúrgico de alta complejidad

Uterine fibroids are the most recurrent pelvic tumor in women, these are derived from myometrial smooth muscle cells and can be located in any of the three main uterine components: fundus, body and neck (cervix). However, they represent only 5% of all uterine fibroids. A case of a cervical uterine fibroid is presented with the purpose of reviewing the clinical manifestations, and its therapeutic management, with a health history, who attends the gynecology service due to an increase in the volume of the abdomen, progressive, approximately three years of evolution, accompanied by genital bleeding and voiding difficulty. A tumor dependent on the uterus (cervix) is diagnosed and in the transoperative period a large tumor with loss in the pelvic anatomy is confirmed. The ligation of hypogastric arteries in large cervical and giant fibroids is an effective therapeutic resource to minimize bleeding and its complications. Myomatous tumor of the cervix should be taken into account in the differential diagnosis of abdominopelvic masses. From the anatomical point of view, hemorrhagic complications are frequent, so total abdominal hysterectomy with ligation of the hypogastric arteries is a good resource to apply.

Proteomic and miRNA Profiles of Exosomes Derived from Myometrial Tissue in Laboring Women.

Myometrial contraction is essential for successful delivery. Recent studies have highlighted the vital roles of tissue-derived exosomes in disease diagnostic, prognostic, and therapeutic applications; however, the characteristics of uterine myometrium-derived exosomes are unclear. Here, we successfully isolated exosomes from myometrial tissues, human myometrial smooth muscle cells (HMSMCs), and human umbilical vein endothelial cells (HUVECs), then performed quantitative liquid chromatography-tandem mass spectrometry and miRNA sequencing to investigate the cargo of the exosomes. Fifty-two proteins and five miRNAs were differentially expressed (DE) in term non-labor and term labor myometrium-derived exosomes. Among them, seven proteins (SERPINE1, THBS1, MGAT1, VIM, FGB, FGG, and VWF) were differentially expressed both in the myometrial exosomes and tissues, three miRNAs (miR-363-3p, miR-203a-3p, and miR-205-5p) target 13 DE genes. The top three miRNA derived from HMSMCs (miR-125b-1-3p, miR-337-5p, and miR-503-5p) and HUVECs (miR-663a, miR-4463, and miR-3622a-5p) were identified. Two proteins, GJA1 and SLC39A14, exist in female blood exosomes and are highly expressed in HMSMCs exosomes, are also upregulated in the laboring myometrium, which verified increased in laboring blood samples, might be novel potential biomarkers for myometrial activation. The proteomic and miRNA profile of exosomes derived from laboring myometrium revealed some molecules in the exosomes that affect the intercellular communication and the function of the myometrium.